A. Field of the Invention
The present invention relates to a method and apparatus for separating portions of liquid in a vessel. Specifically, the invention relates to a method and apparatus for separating portions of a liquid density gradient where layers or zones of materials are produced during the course of centrifugal separation of cellular and/or subcellular material. The present invention is applicable to cell separation and subcellular fractionation and analysis, and to density gradient methods used in molecular biology, in polymer chemistry, in physical chemistry, and in the characterization of physical particles generally. The present invention further relates to an automated method and apparatus for recovering fractions from multiple density gradients in parallel using floats that float on the surface of the gradient.
B. Description of the Related Art
The publications and other materials used herein to illuminate the background of the invention and to provide details respecting the practice are incorporated herein by reference, and for convenience are respectively grouped in the appended List of References and are incorporated by reference in their entirety.
Density gradient centrifugation has been used to separate biological particles on the basis of, for instance, sedimentation rate or banding density. Continuous gradients may be formed either using simple manual techniques, or by using gradient formers, of which a variety have been described (Boch and Ling). In other methods, step gradients may be made initially, and used directly in simplified procedures, or they may be linearized by diffusion, or mechanically by slowly turning the tubes in an angled position. Samples of material, such as cellular material, are placed in a centrifuge tube above such a gradient, subjected to centrifuging in a centrifuge for a predetermined time period and then removed from the centrifuge. The centrifuging causes specific portions or fractions of the cellular material to move to specific portions of the gradient.
If dyes or other observable indicators are present, visual inspection may reveal separate zones or layers of material. The various zones or layers may also be made visible by scattered or reflected light, by light absorbance, or by fluorescence to guide recovery. Once identification of separations is made, one or more zones or fractions of the gradient may be removed for further analysis. Gradients may be removed or recovered by pumping the desired portion of the gradient out of the centrifuge tube through a small tube extending to the bottom of the centrifuge tube. Alternatively, the gradients can be removed by displacing the gradient with a denser fluid introduced to the bottom, with the gradient then collected through a tight-fitting inverted funnel at the top. The gradient may pass through a flow cell in a calorimeter, spectrophotometer, turbidometer or fluorometer to determine the location of particle bands. The simplest method for collecting a gradient is by puncturing a hole in the bottom of a plastic centrifuge tube, and allowing the gradient to drip out, in which case, the drops may be collected individually or in groups.
In zonal centrifuges, gradients are unloaded while the rotor is spinning, and centrifugal force maintains the order of zones. This technique is not readily adaptable to recovering gradients from individual centrifuge tubes.
To attain reproducible precision rate zonal centrifugation or precision isopycnic centrifugation done in multiple-parallel gradients, gradients must be unloaded in a consistent identical manner, with as little loss of resolution as possible. Current unloading methods do not provide a reliable means for processing large numbers of gradients in a reliable, reproducible manner. Therefore, there is a need for a simple method for unloading identical gradients, and for recovering from them identical fractions.
One object of the present invention is to provide a simple and reliable means for unloading individual layers or zones from liquid density gradients.
In accordance with one aspect of the present invention, a float is formed with a concave upper surface thereby defining a well. The float is adapted for insertion into a vessel such as a centrifuge tube having a density gradient therein. The float may be pushed downward in the tube to allow a portion of the density gradient to spill over an upper edge of the float, thereby allowing a desired portion of the density gradient to spill over into the well. A pipetter may then be used to remove the separated portion of the density gradient now located in the well. By repeatedly pushing the float successively lower into the tube, various portions of the density gradient may be easily separated and removed.
The float of the present invention may be used in any of a variety of applications. For instance, the float may be used in manually conducted separation procedures where a technician manually pushes the float down in order to capture a portion of a gradient in the well, then remove the separated portion of the gradient with a pipetter or syringe.
The float of the present invention may form part of an automated system where a remote controlled pipetter is lowered into a tube to both push the float down, and to capture a portion of a gradient. The automated system may include an optical reader to identify zones or layers of the gradient in the tube in order to determine the movement of the float necessary to capture a desired portion of the gradient. Alternatively, the automated system may be programmed to lower the float to a predetermined depth thereby capturing a predetermined portion of a gradient for removal.
The float of the present invention may also form part of an automated system in which samples isolated by the float are expelled from the centrifuge by brief application of air pressure. In this version the pipette tube is stationary, and the centrifuge tube, float, an airtight cap and means for holding the cap in place comprise a unit which is moved vertically at suitable intervals by a stepping motor. The stationary pipette enters the cap through a small O-ring. Tubular means for conduction of air under pressure through the cap are provided, together with means for moving a series of collecting tubes to receive the expelled fractions.
The float of the present invention and the automated fractionation and recovery systems may further be reduplicated to form an array of recovery systems such that a set of identical or comparable gradients may be unloaded in parallel.
The float of the present invention may also be modified through the inclusion of a circumferential groove in the upper cavity of the float so that it may be easily grasped by a gripper which can serve to insert the float before centrifugation, and remove it after the gradient zones have been recovered.
A float of the instant invention can be configured of a particular material or of particular materials so to have a particular density. That also can be achieved by having particular coatings on the float, having the float contain a ballast, by having a hollow float containing a material or materials of particular density and so on. Moreover, the float need not be a solid but can be perforated, for example by a tube or tubes, or can be porous to enable passage of fluids therethrough.